Normalization of nCounter Gene Expression Data Alters Molecular Diagnostics in Kidney Transplantation

The authors aimed to determine how different normalization methods for nCounter gene expression data affect the diagnostic performance of molecular profiling in kidney transplantation, specifically for antibody-mediated rejection (AMR) and T cell-mediated rejection (TCMR). They evaluated ten normalization techniques across multiple cohorts and found that while most methods improved gene count stability and diagnostic accuracy, complex methods like RCRNorm and RUVSeq negatively impacted performance. Ultimately, simpler normalization approaches consistently provided better outcomes for B-HOT-based diagnostics.